Tensile Stress Up-regulate The Expression Of β-catenin Signalling And Chondrogenic Differentiation Related Transcription Factors In Cells Derived From The Human Ossification Of Ligamentum Flavum

IntroductionThe etiology of the OLF (ossification of ligamentum flavum) has been investigated extensively in various fields, including clinical, radiological, and biological aspects. However the exact mechanism of ligament ossification remains poorly understood although the importance of genetic and biological factors has been acknowledged.In the histological point of view, the progression in ossification of spinal ligament was closely correlated to the endochondral ossification. Histological studies of OLF shows marked hypertrophy chondrocyte-like cells at the front of ossification site. Modulating mechanism of ossification in OLF may be similar to endochondral ossification that occurred in skeletogenesis and growth plate. Resent reports revealed that Wnt/β-Catenin pathway play a key role in endochondral ossification through modulating the chondrocyte maturation and hypertrophy during skeletogenesis and growth plate development. Whether this signaling pathway also play a role in the process of ossification of spinal ligament is still unknown.Local environment factors like abnormal mechanical force could also contribute to spinal ligament ossification. Animals experiment show repetitive stress can induce cartilage formation in spinal ligament, this suggest that repetitive tensile stress to the spinal ligaments is one of the important causes of ligament ossification in the spine. Clinical study also showed the progression of ossification of the posterior longitudinal ligament was highly correlated with abnormal strain distribution in the intervertebral discs. It also have been suggested that mechanical stretch can affect ligament cells through different pathways, included TGF-β1,osteopontin, PGI2/cAMP system, Runx2 and BMP-2 under the mechanical stress.Resent findings indicate a stimulating role of Wnt/(3-Catenin signaling in cellular mechanotransduction. Studies showed that Wnt/beta-catenin signaling is a normal physiological response to load and activation of the Wnt/beta-catenin pathway also enhances the sensitivity of osteoblasts/osteocytes to mechanical loading.The present study was designed to determine the role of Wnt/β-Catenin pathway in mechanotransduction and modulation of chondrocyte differentiation related transcription factors using ligamentum flavum tissue samples and cultured ligamentum flavum cells under cycle tensile stress. Specifically, we focused on the expression and localisation of signals that modulate the properties of fibroblasts and chondrocytes during the process of ossification.Patients and MethodsA total of 49 patients who presented with progressive symptoms and signs of myelopathy and radiological evidence of OLF,8 patient for cell culture underwent posterior decompressive surgery. Obtained from 8 patients were used as the control. Another 6 patient samples for cell culture and mechanical stress.Cell CultureThe ligaments were harvested aseptically during surgery and the surrounding tissue was removed carefully under a dissecting microscope. The ligaments were extirpated carefully from the non-ossified areas to avoid contamination with osteogenic cells. The subcultured cells were observed under a phase-contrast microscope before they had retained the morphologic characteristics.Assessment of cell survival and cell DamageTo determine possible cellular damage due to tensile stress, cell survival was examined by manual cell counting at 0,6,12,24 hours after the application of tensile stress. Live/Dead(?) Viability/Cytotoxicity Assay Kit Assessments were performed as described previously by our group. The cell survival rate was calculated.In vitro application of Mechanical stress to cultured cellsThe cell stretching device used in this study was a FX-3000 the device consisted of a computer-controlled vacuum unit, a culture well plate with a flexible-polystyrene culture well bottom coated with typeⅠcollagen, and another culture well plate with a non-deformable culture well bottom made of the same materials as FlexⅠ. The culture plates consisted of 6-well with the hydrophilic, flexible bottoms. After 3 days cell seeding, cells were subjected to cyclic stretch stress for 24 hours. The cell remained attached to the substratum during elongation. The cells were observed morphologically, quantification of mRNA expression of transcription factors, and immunoreactivity were conducted at 0,6,12,24 hours after application of tensile stress.Real-time Polymerase Chain Reaction AnalysisRT-PCR was used for semi-quantitative analysis of the relative mRNA expression levels of transcriptional factors (repeated three times for sample), using cultured ligamentum flavum cells from the ossified (mixed 6 case samples) and non-ossified groups (mixed 6 control samples). The cultured cells on each plate were disrupted in a lysis buffer containingβ-mercaptoethanol and the total RNA was purified using RNAiso. The expression levels of the target genes were estimated relative to that of GAPDHHistopathological & Immunohistochemical StainingThe resected OLF plaque together with the surrounding ligament and ligamentous enthesis were embedded in paraffin using standard procedures. Serial 4-μm thick sagittal sections of the OLF-ligament-enthesis complex were prepared for H&E, Safranin O staining, and elastic fiber EVG staining.For immunohistochemical staining, serial 4μm-thick sections were prepared from the paraffin-embedded specimens, deparaffinized with xylene and treated with ethanol. After washing with water, the intrinsic peroxidase was blocked with 0.3% H2O2-methanol solution at 20℃for 10 minutes and washed with phosphate buffered saline (PBS;pH 7.4). The sections were irradiated three times in a polypropylene side-holder with a cap filled with PBS over a period of 5 minutes, using a microwave oven (500 w, ER-245, Toshiba, Tokyo). The sections were then reacted with BLOCKING solution at 20℃for 10 minutes. This was followed by reaction with the following primary antibodies at 4℃overnight:rabbit polyclonal anti-Sox9; rabbit polyclonal anti-Osterix; mouse antiβ-Catenin; goat polyclonal anti-OPN; rabbit polyclonal anti RUNX2 antibody; mouse monoclonal anti TGF-βantibody; DakoCytomation EnVision+(?) system and DAB was used to detact the immuno-reaction. Nuclear counterstaining was carried out with haematoxylin.ResultEffects of cyclic tensile stress on cell morphology and cell survivalOn confluence both OLF and non-OLF cells exhibited a fibroblast-like, spindle-shaped appearance. Morphological change was seen in some OLF cells. A few oval cells existed among an abundance of spindle cells. The proportional and phenotypic characteristics did not differ from each specimen or each passage of cells.The attached OLF cells showed a viability of 92.4±2.35% and 90.4%±2.90% in 24h-stretched and non-stretched regimens, respectively (Fig.2). On each time, the viability of stretched OLF cells did not differ from that of FL cells (Student's t-test p> 0.05). All of 6 OLF cells were used in the following experiments; they showed similar responses to mechanical stress as described blew.Histopathological and Immunohistochemical FindingsThe H&E staining show the similar calcification pattern to the cases our previously reported. The safranin O staining confirmed that there are chondrocytes at front of the calcification front (Fig.). The chondrocytes present in the ossification front were immunopositive for, Sox9, Osterix, OPN and TGF-β, but negative for wnt8a andβ-catenin;the immunoreactivity ofβ-catenin was strictly limited to the premature chondrocytes in the fibrocartilage area, whereas the hypertrophic chondrocytes close to the calcification front were negative forβ-catenin (Fig.). On the other hand,β-catenin and Sox9 expression was significant in mesenchymal cells present around the ossification front, where the ligamentous matrix showed degenerative changes in these patients. Runx2 expression localized osteoblasts in ossification site. Immunopositive for Oxterix was also noted in premature chondrocytes, mature chondrocytes, and in particular, the mesenchymal cells near the ossification front were strongly positive for Osterix. Furthermore, numerous Osterix-positive hypertrophic chondrocytes were seen around the calcification front.PCRThe relative mRNA expression levels of six transcriptional factors in cultured ligamentum flavum cells from OLF and non-ossified samples. Before the mechanical stimulating, mRNA expression levels of Runx2,, OPN and TGF-(3 (relative to the GAPDH expression level) in the ossified group were high than those in non-ossified group. Then mRNA expression levels of Osterix and Sox were not significantly different between two groups. The relative mRNA expression levels ofβ-Catenin, Osterix and TGF-βin OLF group increased with time and arrived highest points at 24h, and significantly higher than those in the control group (p<0.05, each). However, the mRNA expression levels of Sox9 in non ossified group also increased significantly after stretching 6h.DiscussionPrevious researches mainly focused on modulating mechanism of osteoblast differentiation. However, progression in understanding the process of endochondral ossification in growth plate demonstrated that the regulating the chondrocytes differentiation and maturation is the most important in endochondral ossification process.Through triggering changes in genes expression by chondrocyte at the growth plate, systematic hormones and local growth factors could regulate ossification precisely. Ossification process, such as new vascular invasion and osteoblast migration, are secondary stage to chondrocyte hypertrophy. Furthermore hypertrophic chondrocytes is also required for osteoblast maturation and endochondral bone formation as secreting factors required for osteoblasts differentiation, maturation, and migration under the influence of growth factors or transcriptional factors.Recently studies demonstrated that Wnt/β-Catenin pathway play a key role in endochondral ossification, Wnt/beta-catenin signaling is a critical regulator of the rate of chondrocyte differentiation. However it is still unknown whether Wnt/β-Catenin involved in OLF.Wnt/beta-catenin signaling regulates chondrocyte phenotype, maturation, and function in a developmentally regulated manner, and regulated action by this pathway is critical for growth plate organization, cartilage boundary definition, and endochondral ossification. Up-regulation of beta-catenin could accelerat the process of endochondral ossification including chondrocyte maturation and osteoblast differentiation. Guo reported the wnt/beta-catenin signaling regulates initiation of chondrocyte hypertrophy and also controls final maturation of hypertrophic chondrocytes. Since similar controlling mechanisms of chondrocyte proliferation and differentiation may be employed by adult mesenchymal progenitor cells during ossification in FL.Our result show the immunoreactivity ofβ-catenin was strictly limited to the premature chondrocytes in the fibrocartilage area, whereas the hypertrophic chondrocytes close to the calcification front were negative forβ-catenin. This indicatesβ-Catenin maybe be involved in early stage of chondrocyte-like cell hypertrophy.However,β-Catenin pathway was showed different roles in embryonic skeletogenesis and ectopic bone formation. In vivo ectopic bone formation, upgrade B-catenin is required for both chondrogenesis and osteogenesis. It's in contrast to study of embryonic skeletogenesis, in whichβ-catenin is required for osteogenesis but not chondrogenesis. Negative effects of Wnt signaling on osteoblast differentiation and mineralization have been described as well. Our result suggests that theβ-Catenin signaling is critical for hypertrophy of chondrocyte-like cells in OLF, which is similar to the role in growth plate but contrast to embryonic skeletogenesis.Mechanical force also could have an important role in development of OLF. Some cytokines and pathway have been studied for possible role in mechanical stress induce the progression of spinal ligament ossification, such as prostaglandin 12, Runx2 and TGF-beta. Recent studies support that Wnt/beta-catenin signaling is a normal physiological response to load and that activation of the Wnt/beta-catenin pathway enhances the sensitivity of osteoblasts/osteocytes to mechanical loading. Sen report the mechanical strain inhibits adipogenesis in mesenchymal stem cells by stimulating a durable beta-catenin signal. It is likely that there have some association between the mechanical force and beta-catenin in OLF.Interaction between theβ-catenin signaling and chondrogenic differentiation related transcription factors pathways is likely to play an important role during ossification process in ligamentum flavum. The beta-catenin signaling pathway maybe mediates other transcription factors. Our results showβ-catenin increase Runx2 and sox9 expression.Osteoblasts and chondrocytes are thought to differentiate from a common mesenchymal precursor, the osteo-chondroprogenitor. Although numerous transcription factors involved in chondrocyte and osteoblast differentiation have been identified, little is known about the signals controlling lineage decisions of the two cell types. Runx2 and Sox9 were considered two critical factors in early stage of chondrocyte differentiation.As Sox9 is expressed in all chondrocytes at all developmental stages, the regulation of Sox9 activity is at least one of the underlying mechanisms for the opposite activities of canonical Wnt and Pthrp signaling in regulating chondrocyte hypertrophy. Osterix had been proved a key target for mechanical signals in human thoracic ligament flavum cellsOut result show that the chondrogenic potential of mechanical stretching force stimulation was confirmed by an increase in expression level of chondrogenic differentiation markers such as Sox9 and Runx2 after 24h stretching, suggesting that they could have a role in the pathophysiology of the heterotopic ossification of ligamentum flavum.The present study indicates a previously unrecognized role for the beta-catenin signaling pathway in process of ossification of the ligamentum flavum. It may be a new target for preventing the progression of OLF. But more studies need to been done to elucidate the up and stream signaling pathway.